The transforming growth factor-beta (TGF-.beta.) superfamily of cytokines regulate a diverse array of physiologic functions including cell proliferation and growth, cell migration, differentiation, development and apoptosis. This large family includes the TGF-.beta.s, activins, and bone-morphogenic proteins (BMPs) and each subgroup initiates a unique signaling cascade activated by ligand-induced serine/threonine kinase receptor complex formation (Wrana, Miner. Electrolyte Metab., 1998, 24, 120-130). These complexes, once formed, recruit and phosphorylate members of a family of cytosolic proteins, known as Smads. Smads exist as monomers in unstimulated cells but homo- or heterodimerize and translocate to the nucleus activating target gene transcription upon ligand binding. Smads, therefore, connect the pathway of TGF-.beta. signaling from the cell membrane to the nucleus.
To date, nine vertebrate Smads have been identified and these have been divided into subgroups based on their functional role in various pathways. Smad1, 5, and MADH6, which is 80% homologous to Smad1, all mediate signal transduction from BMPs while Smad2 and 3 mediate signal transduction from TGF-.beta.s and activins. Collectively, these Smads are known as the pathway-restricted Smads and can form homo or heterodimers. Smad4 has been shown to be a shared hetero-oligomerization partner to the pathway-restricted Smads and is known as the common mediator. The last two members of the family, Smad6 and 7, act to inhibit the Smad signaling cascades often by forming unproductive dimers with other Smads and are therefore classified as antagonistic Smads (Heldin et al., Nature, 1997, 390, 465-471; Kretzschmar and Massague, Curr. Opin. Genet. Dev., 1998, 8, 103-111).
Smad5 (also known as MADH5, Dwarfin-C and JV5-1) is a member of the subgroup of Smad family transcription factors which are regulated by, and mediators of, bone morphogenic proteins (BMPs). BMPs play important roles in vertebrate organogenesis and embryogenesis. Smad5 was first isolated during studies designed to identify proteins that are involved in osteoblastic differentiation of pluripotent mesenchymal precursor cells (Nishimura et al., J. Biol. Chem., 1998, 273, 1872-1879). In these studies, Smad5 was shown to be activated by BMP-2 through the BMP type Ia or Ib receptors and then to transduce signals to the nucleus by associating with the common mediator Smad4.
The role of Smad5 in muscle and bone development has been the primary focus of the limited studies of Smad5 to date. In studies of BMP signaling using C2C12 myoblast cells, Smad5 was shown to be involved in convergent signaling pathways that inhibit myogenic differentiation and induce osteoblast differentiation (Nishimura et al., J. Biol. Chem., 1998, 273, 1872-1879; Yamamoto et al., Biochem. Biophys. Res. Commun., 1997, 238, 574-580).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of Smad5 and to date, strategies aimed at inhibiting and/or investigating Smad5 function have involved the use of mutations of the protein. Overexpression of a point-mutated form of Smad5 or a deletion mutant of Smad4 was shown to block the Smad5 mediated signaling cascade that leads to osteoblastic differentiation suggesting that the Smad5:Smad4 complex is a necessary component of this pathway (Nishimura et al., J. Biol. Chem., 1998, 273, 1872-1879).
In light of the lack of inhibitory strategies targeting Smad5, there remains a long felt need for additional agents capable of effectively inhibiting Smad5 function. Therefore, antisense oligonucleotides may provide a promising new pharmaceutical tool for the effective and specific modulation of Smad5 expression.